Eukaryotic motor proteins utilize the cytoskeleton as roadways to transport a variety of intracellular cargo across relatively vast distances. The reaction of ATP with water to produce ADP and inorganic phosphate is the central and primary step in motor proteins that initiates critical cellular functions, including chromosome segregation during mitosis, gene replication, transcription, and transport of vesicles and organelles. These nanomotors all function by consuming this energy and coupling the chemical intermediates to a series of conformational changes that propel net celular motion. For kinesins, defects in this catalytic reaction and its chemo-mechanical transduction are linked to cancer, developmental errors, myopathies, and neurodegenerative conditions in humans. Although a complex sequence of intermediate states during ATP hydrolysis and mechanotransduction is predicted, only a small subset of states has been validated by direct structural observation. Thus, current empirical evidence of atomic-level interactions is not sufficient to assess the complete range of chemical steps in biological ATP hydrolysis and its partnered energy transduction. Recent data from our laboratories has captured key catalytic intermediates that resolve extant questions regarding mechanism and provide a new framework for further progress. Building upon these findings, our principal goal is to define novel roles for proton transfer and hydrogen bonding for ATP hydrolysis and chemo-mechanical coupling in the human Kinesin-5 protein, essential for mitosis and a target for cancer therapeutics. Questions to be tested are the identity of the chemical player in the first step of ATP hydrolysis, whether loops flanking the active site undergo conformational changes in the enzyme transition state, and if the central 2-sheet has a role in transducing information from the active site to other sites. The significance of the anticipated answers is twofold. As chemistry and motion are tautologically linked, this information is required to illuminate molecular mechanisms of motion in nanomotors, which are currently unclear from these deficits. Moreover, garnered experimental evidence will allow rational design of anti-cancer drugs directed against human Kinesin-5. PUBLIC HEALTH RELEVANCE: Kinesin motors are the smallest moving machines known in biology, and humans have approximately 50 different kinds of kinesin motors. Understanding the key reaction in obtaining energy to operate and move can lead to control of not only individual, cellular processes, but also specific cell types. The information garnered from this proposal may offer clinical power over human syndromes, such as dividing cancer cells, neurological disorders, and infertility.